In Planta Deletion of DNA Inserts in the Large Intergenic Region of Cauliflower Mosaic Virus DNA

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Over the course of my graduate career in the Oklahoma State University Biochemistry Department my research objectives have evolved. My original objective was to determine the utility of cauliflower mosaic virus (CaMV) DNA as a vehicle to carry inserted DNA sequences into plants for gene targeting experiments. Specifically, my intention was to construct, in plasmid form, one or more infectious, stable CaMV DNAs bearing fragments of the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene coding region. Once stable constructs were obtained, I planned to look for inactivation of the ADH gene in Arabidopsis seeds obtained from plants inoculated and systemically infected with the constructs. I would then examine any plants grown from ADH- seeds for evidence that the observed ADH gene inactivation was due to insertion of CaMV DNA sequences within the gene. I was successful in creating several plasmid CaMV DNAs bearing pieces of ADH DNA. These fragments were added to a portion of the CaMV genome called the large intergenic region (IG) using, as recipient for insertions in the IG, a plasmid CaMV clone constructed by Dr. Melcher. The next step was to inoculate turnips with these plasmids to obtain material for virion inoculation of Arabidopsis. This was necessary because plasmid inoculation of Arabidopsis is difficult and inefficient. However, after inoculating turnips with the constructs I discovered that the ADH inserts were invariably deleted in plants. Given 1) the instability of the CaMV/ADH constructs, 2) time constraints associated with my degree program, and 3) other anticipated difficulties I decided, in consultation with Dr. Melcher, to change research objectives. We decided that I would continue and complete work on a project of his which was both on-going and relevant to my initial results showing instability of ADH gene fragments in the IG. Specifically, the goal of this project was to discern mechanisms governing stability, or instability, of insertions in the I G. This paper contains results of initial experiments, performed by myself and by Dr. Melcher (with the able assistance of his two laboratory technicians, Ann Williams and Mary Schatz), as well as results of experiments and analyses performed by me after I took over the project. Following is a breakdown of specific contributions to the manuscript. Dr. Melcher constructed and determined the infectivities of the following plasmid cloned viral DNAs: pCMLl, pCaMTL( + ), pCaMTL(-), pCaMTL(+)f, and pCML3 through pCML13 (Table 1). He also performed the restriction analyses of viral DNAs recovered from plants infected with these clones(Figs. 4A, 4B, SA, 5B, and 5C) as well as analyses of viral DNAs recovered from plants inoculated with cloned progeny DNAs and passaged viral DNA (Figs. 7 and 8). I constructed pRPs 1(+), 1(-), 5(-), 5(2+), 5(+), 6(+), and pCML5d (Table 1) and performed the corresponding restriction analyses (Fig. 5D, 5E, and 6). I also performed .. additional restriction analyses of viral DNAs recovered from pCML1 and pCaMTL(-) infected plants. These results are not shown in any figure but are included in the results and discussion. Cloning and partial DNA sequencing of individual viral DNAs recovered from selected plants was performed by Dr. Melcher (pCML( + ), pCML3, pCML5) and by myself (pCMLl, pRPl(+)). These results are summarized (Table 2) as are results of dot matrix homology analyses performed by myself. All summaries, conclusions, and speculations in the manuscript are my own. A decision made during the writing was that our findings would be written in the form of one longer manuscript rather than two or more shorter ones. A consequence of this decision is that rather than having two or more thesis chapters consisting of separate manuscripts to be submitted for publication, I have only one. Thus, I am presenting the parts of the manuscript as individual chapters. The manuscript is intended to be complete and ready, except for minor formatting details, to be submitted to the journal Virology under the authorship of myself and Dr. Melcher. As journal article introductions are generally brief, I am including in this preface additional general information about CaMV which will assist the reader in understanding the manuscript. CaMV Background Information Biology and Structure of CaMV. Cauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of only two known groups of plant viruses having, for a genome, double-stranded DNA (see review by Shepherd (1989) for information not otherwise attributed). CaMV infects many species of the Brassicaceae (including Arabidopsis thaliana and Brassica rapa) and some isolates infect some members of the Solanaceae. CaMV-infected plants show symptoms of various types and severities depending on the infecting isolate and host species. Possible symptoms include leaf mosaics or mottles, distortion of leaves, and stunting. The virus is transmitted by aphids in nature and can also be transmitted by mechanical inoculation. The bulk of CaMV virions in infected cells are localized in large cytoplasmic proteinaceous inclusion bodies which serve as the site of virus assembly (Marsh et al., 1985). The virions themselves are 50 nm icosahedral particles consisting of approximately 84% protein and 16% DNA. The double-strandedness of DNA found in virions is interrupted by two or three (depending on the isolate) site-specific gaps. One of these gaps is invariably found in the minus DNA strand and by convention defines position 1 in the clockwise numbering of the plus strand DNA sequence (12 o'clock position, Fig. 1A). Genetic Organization of CaMV and ORF Products. The DNA is approximately 8 kilobasepairs (kbp) and contains eight large open reading frames (ORFs), all in the plus strand (see Fig. 1A). ORFS I through V are tightly packed, in some cases abutting or overlapping. ORF VI is separated from ORFs V and VII by intergenic regions of 100 and 700 basepairs (bp ), respectively. ORF VIII (not shown) is out of frame with and overlaps the 3' end of ORF IV. Functions have been shown or surmised for most of these ORFs. ORF I encodes a protein similar in sequence, and possibly function, to the cell-to-cell movement proteins of tobacco mosaic virus and other viruses (Hull et al., 1986; Melcher, 1990). ORF II encodes a protein required for aphid transmission (Armour et al., 1983; Woolston et al., 1983). ORF III encodes a protein which has DNA binding activity and may be a structural component of the virion (Mesnard et al., 1990). ORF IV encodes the coat protein precursor (Daubert et al., 1982). All evidence indicates ORF V encodes the viral replicase, in this case an RNA-dependent DNA polymerase (reverse transcriptase) (Takatsuji et al., 1986). ORF VI encodes the inclusion body protein (Xiong et al., 1982) which forms the inclusion body matrix. This ORF also contains symptom (Daubert et al., 1983) and host-range determinants (Schoelz et al., 1986) and has further importance as discussed later. ORFs VII (Dixon and Hohn, 1984) and VIII (Schultze et al., 1990) are dispensable for infection and may be not expressed in infected plants. CaMV Infection Cycle. Entry into the host cell is accomplished either by aphid feeding or mechanical inoculation. After uncoating, viral DNA in the nucleus has its gaps repaired by host mechanisms. The result of repair is a supercoiled CaMV minichromosome which is transcribed by host RNA polymerase II (Olszewski et al., 1982). Two major polyadenylated RNA species are produced, both by transcription of the CaMV minus-strand [These early events are summarized in Hull and Covey, (1985)]. The smaller of these RNAs, the 19S, spans ORF VI and serves as a messenger RNA for its expression (Covey and Hull, 1981). The larger RNA, the 35S, encompasses the entire genome and its 5'-end maps to a location 100 bp downstream of ORF VI (Guilley et al., 1982). The 3'-termini of both transcripts map to the same position 180 bp downstream of the 5'-end of the 35S RNA. In the 35S RNA this results in a 180 bp terminal redundancy. The function of the 35S RNA is discussed below. CaMV is a "retroid element". This designation encompasses mammalian retroviruses and other agents which replicate their nucleic acid using reverse transcriptase. Specifically, CaMV DNA replication proceeds with reverse transcription of the 35S RNA by CaMV-encoded reverse transcriptase to produce a minus-strand DNA (see review by Hohn et al., ( 1985) ). Priming of reverse transcription is provided by the binding of the 3' portion of a methionine initiator tRNA to a complementary sequence in the 35S RNA located 600 bp downstream of its 5' terminus (5' end of tRNA binding site corresponds to position 1). Reverse transcription proceeds counterclockwise to the 5' end of the 35S RNA where the reverse transcriptase and the nascent minus-strand DNA must switch templates to the 3'-end of the same, or another, 35S RNA molecule. After this obligatory switch, reverse transcription proceeds full-circle to produce a full length, but covalently open, circular DNA strand. After, or concurrently with, minus-strand DNA synthesis, the RNA template is degraded (by an RN ase H activity associated with CaMV reverse transcriptase) except for one or two short stretches which map in ORF V and, in some isolates, ORF II. The RNA in these regions of residual DNA/RNA basepairing serves as primer(s) for synthesis, by reverse transcriptase, of the plus-strand DNA. A second obligatory template switch occurs when reverse transcriptase, now synthesizing in the clockwise direction, encounters the gap located at position 1 in the covalent structure of the minus-strand. After this template switch, further plus-strand DNA synthesis completes the CaMV circle, terminating just downstream of the point(s) at which synthesis was primed. The net result is a CaMV DNA molecule with one or two site-specific gaps in the plusstrand DNA and one in the minus strand. This DNA is packaged in virions. CaMV Translation. As mentioned, the 19S RNA serves as an mRNA for ORF VI. Translation of ORFs I through V is somewhat mysterious, but it is generally believed that these five ORFs are translated utilizing the 35S RNA as a polycistronic mRNA. In this unusual mode of eukaryotic translation, referred to as the "relay race", translation of the 35S proceeds from one ORF to the next in the 3' direction without dissociation of the ribosome from the template (Dixon eta/., 1983). Relay-race translation is consistent with, and dependent upon, the tight packing observed for the first five ORFs. Efficient expression of ORFs I to V is also dependent upon a trans-acting effect of the ORF VI product, probably the protein (Bonneville eta/., 1989; Gowda eta/., 1989). A large stemloop structure, proposed for the untranslated, 5'-most 600 nucleotides of the 35S RNA, may be important in determining the balance between translation and reverse transcription of this transcript (Fuetterer et a/., 1990). The information above is intended as a source of background know ledge for those unfamiliar with CaMV molecular biology. The manuscript itself deals with the stability of DNA insertions in the CaMV IG. Background information specifically related to this topic is found in the introduction.